The long-term goal of this project is to dissect the sequence of conformational changes of pre-messenger RNAs (pre-mRNAs) and the surrounding spliceosome active site that together constitute the splicing mechanism. The spliceosome is the multi-megadalton RNA-protein complex that in all eukaryotes catalyzes the removal of introns and the ligation of exons during splicing of pre-mRNAs. In humans, ~95% of all pre-mRNAs undergo alternative splicing, which allows for the dynamic expression of various protein isoforms from a single gene through cell- and tissue-specific networks of regulated splicing events. It is estimated that up to 50% of all mutations leading to human disease act through disrupting the splicing code. The budding yeast Saccharomyces cerevisiae has long provided the preferred model system for dissecting the mechanism of splicing, due to the availability of unique genetic and biochemical manipulation tools and an increasing amount of static structural information. Despite 30 years of study, however, there remain substantial gaps in our understanding of the timing and coordination of the conformational rearrangements associated with yeast splicing. To address this challenge, we have developed multiple single molecule fluorescence resonance energy transfer (smFRET) assays and other tools that have begun to dissect the pre-mRNA conformational changes of splicing. In addition, we have adopted modern RNA deep-sequencing techniques to interrogate the identities and secondary structures at single-nucleotide resolution of all pre-mRNAs found in the Bact complex. We now propose to leverage these advances to dissect incisively the mechanism of splicing. In Specific Aim 1, we will test the hypothesis that kinetic competition between the first chemical step of splicing and the displacement of the `pawl' Cwc25 by the RNA helicase Prp16 determines whether substrates with non-ideal branchpoint sequences are spliced or instead rejected through proofreading. We will also test the hypothesis that ? in analogy to our results on helicase Prp2 ? Prp16 action leads to biased Brownian ratcheting. In Specific Aim 2, we will follow up on our observation that certain introns exhibit significant secondary structure with the 5' exon much closer to the branchpoint than expected from their sequence distance. We will test the hypothesis that this is a common feature among well- splicing pre-mRNAs by collaborating with sub-contractor Alain Laederach on applying selective 2?-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to the over 250 pre-mRNAs we found in the in vivo isolated spliceosomal Bact and C complexes, enriched in yeast strains with temperature- sensitive mutations in Prp2 and Prp16, respectively. In Specific Aim 3, we will develop Expedited Position- selective Labeling Of RNA (ExPLOR) to internally label the spliceosomal U5 small nuclear RNA (snRNA) near Loop 1 to test the hypotheses that Prp22 disrupts the U5:pre-mRNA interaction for proofreading before and for disassembly after the second step of splicing. Taken together, our advances will continue to provide tools for dissecting a plethora of RNA-driven biomolecular machines and pave the way for studying alternative splicing.